Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol.

BACKGROUND: In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol. METHODS: Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol. RESULTS: In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol. CONCLUSION: This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol. GENERAL SIGNIFICANCE: The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.

Serum thymidine kinase 1 activity as a prognostic biomarker in dogs with chemotherapy-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is frequently treated with chemotherapy incorporating cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), which induces remission in 80% to 95% of cases. However, not all dogs derive meaningful benefit from CHOP, and prognostic factors for dogs with DLBCL are poorly defined. Serum thymidine kinase 1 (TK1) activity, a marker of tumour cell proliferation, has shown promising initial results as a prognostic biomarker in dogs with multicentric lymphomas. The purpose of this study was to determine if baseline serum TK1 activity is associated with clinical outcome in dogs with CHOP-treated DLBCL. Baseline serum TK1 activity was measured in banked sera from 98 dogs with CHOP-treated DLBCL using a commercially available ELISA kit. Data on other potential prognostic factors were abstracted retrospectively from electronic medical records. Multivariable statistical methods were used to identify associations between TK1 and other potential prognostic factors with progression-free survival (PFS) and attainment of complete remission. TK1 activity at baseline was not associated with PFS (p = .299) or attainment of complete remission (p = .910) following CHOP chemotherapy. Of the other prognostic factors analysed, only purebred (vs. mixed breed) status (HR 8.81, 95% CI 1.68-46.30, p = .010), attainment of complete (vs. partial) remission (HR 0.09, 95% CI 0.02-0.49, p = .006), and baseline serum C-reactive protein concentration (HR 1.19, 95% CI 1.07-1.32, p = .001) were independently associated with PFS. Based on these findings, baseline serum TK1 activity does not appear to be a useful prognostic biomarker in dogs with CHOP-treated DLBCL.

Effects of low-dust forages on dust exposure, airway cytology, and plasma omega-3 concentrations in Thoroughbred racehorses: A randomized clinical trial.

BACKGROUND: Racehorses commonly develop evidence of mild asthma in response to dust exposure. Diets deficient in omega-3 polyunsaturated fatty acids (Ω-3) might exacerbate this response. HYPOTHESIS: To compare dust exposure, bronchoalveolar lavage fluid (BALF) cytology, and plasma Ω-3 and specialized pro-resolving mediators (SPM) concentrations amongst racehorses fed dry hay, steamed hay, and haylage. ANIMALS: Forty-three Thoroughbred racehorses. METHODS: Prospective clinical trial. Horses were randomly assigned to be fed dry hay, steamed hay, or haylage for 6 weeks. Measures of exposure to dust in the breathing zone were obtained twice. At baseline, week-3, and week-6, BALF cytology was examined. Plasma lipid profiles and plasma SPM concentrations were examined at baseline and week 6. Generalized linear mixed models examined the effect of forage upon dust exposure, BALF cytology, Ω-3, and SPM concentrations. RESULTS: Respirable dust was significantly higher for horses fed hay (least-square mean ± s.e.m. 0.081 ± 0.007 mg/m3 ) when compared with steamed hay (0.056 ± 0.005 mg/m3 , P = .01) or haylage (0.053 ± 0.005 mg/m3 , P < .01). At week 6, BALF neutrophil proportions in horses eating haylage (3.0% ± 0.6%) were significantly lower compared with baseline (5.1 ± 0.7, P = .04) and horses eating hay (6.3% ± 0.8%, P < .01). Plasma eicosapentaenoic acid to arachidonic acid ratios were higher in horses eating haylage for 6 weeks (0.51 ± 0.07) when compared with baseline (0.34 ± 0.05, P < .01) and horses eating steamed (0.24 ± 0.02, P < .01) or dry hay (0.25 ± 0.03, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Steamed hay and haylage reduce dust exposure compared with dry hay, but only haylage increased the ratio of anti-inflammatory to pro-inflammatory lipids while reducing BAL neutrophil proportions within 6 weeks.

TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous lipopolysaccharide infusion.

Gram-negative bacterial septicemia is mediated through binding of lipopolysaccharide (LPS) to mammalian toll-like receptor protein 4 (TLR4). TLR4 and its cognate protein, myeloid differentiation factor 2 (MD2) form a heterodimeric complex after binding LPS. This complex induces a cascade of reactions that results in increased proinflammatory cytokine gene expression, including TNFα, which leads to activation of innate immunity. In horses, the immune response to LPS varies widely. To determine if this variation is due to differences in TLR4 or MD2, DNA from 15 healthy adult horses with different TNFα dynamics after experimental intravenous LPS infusion was sequenced across exons of TLR4 and MD2. Haplotypes were constructed for both genes using all identified variants. Four haplotypes were observed for each gene. No significant associations were found between either TNFα baseline concentrations or response to LPS and haplotype; however, there was a significant association (P value = 0.0460) between the baseline TNFα concentration and one MD2 missense variant. Three-dimensional structures of the equine TLR4-MD2-LPS complex were built according to haplotype combinations observed in the study horses, and the implications of missense variants on LPS binding were modeled. Although the sample size was small, there was no evidence that variation in TLR4 or MD2 explains the variability in TNFα response observed after LPS exposure in horses.

Interleukin-6 and thrombopoietin concentrations in dogs with carcinoma with and without thrombocytosis.

BACKGROUND: Carcinoma-associated thrombocytosis involves tumor production of mediators such as interleukin-6 (IL-6) and thrombopoietin (TPO) that increase thrombopoiesis and may play a role in tumor evasion and metastasis. Carcinoma-associated thrombocytosis is described in people, but has not been described in dogs. HYPOTHESIS/OBJECTIVES: Evaluate the concentrations of IL-6 and TPO in dogs diagnosed with carcinoma with or without thrombocytosis. We hypothesized that IL-6 and TPO concentrations would be higher in dogs with carcinoma compared to healthy dogs, and that IL-6 and TPO concentrations would be higher in dogs with carcinoma and thrombocytosis when compared to dogs with carcinoma and normal platelet counts. ANIMALS: One-hundred sixteen dogs: 63 with carcinoma and 53 healthy control dogs. METHODS: Complete blood count was performed in all dogs, and they were stratified for sub-group analysis based on the presence or absence of thrombocytosis (platelet count > 500 103/µL). Serum TPO and IL-6 concentrations were measured by ELISA. Results of selected numeric variables were compared using Wilcoxon rank sum tests for pairwise comparisons. A value of P < .05 was considered significant. RESULTS: Twelve of the dogs with carcinoma (12/63, 19.0%) and none of the healthy control dogs (0%) had thrombocytosis. Thrombopoietin concentrations (median [range]) were significantly higher in dogs with carcinoma when compared to controls (87.42 pg/mL [0 to >600] vs 15.99 pg/mL [0 to >600], P < .001). Interleukin-6 concentrations (median [range]) were not different between dogs with carcinoma and healthy control dogs (9.70 pg/mL [0-181.53] vs 3.03 pg/mL [0-280.77], P = .15). In dogs with carcinoma, the TPO and IL-6 concentrations were not different between dogs with thrombocytosis and dogs with normal platelet count. CONCLUSIONS AND CLINICAL IMPORTANCE: Thrombopoietin concentrations were significantly higher in dogs with carcinoma, regardless of platelet count. Thrombopoietin is likely to be 1 of multiple factors that can impact platelet number, production, and consumption in dogs with carcinoma.

Effects of a single dose of orally administered gabapentin in dogs during a veterinary visit: a double-blinded, placebo-controlled study.

OBJECTIVE: To evaluate the effects of a single dose of orally administered gabapentin in alleviating stress at a veterinary visit in privately owned dogs. Animals: 22 healthy client-owned dogs (1.5 to 8.5 years old) were enrolled in this study. PROCEDURES: Each dog received a 50-mg/kg oral dose of either gabapentin or placebo 2 hours before the beginning of each visit protocol. The dog's behavioral responses were coded from recorded video clips during a 5-minute-long standardized physical examination and pre- and post-physical examination phases. The veterinary technician separately rated each greeting behavior at each visit. Physiological variables during veterinary visits (ie, eye surface temperature and salivary cortisol concentrations) were also compared between the pre- and post-physical examination phases. The owner was queried 24 hours after a visit to determine the incidence of adverse events. RESULTS: The greeting test score, eye surface temperature, and cortisol concentrations did not differ substantially between the gabapentin and placebo treatment groups. Lip licking frequency during the physical examination phase was significantly lower in the gabapentin treatment group than in the placebo group (P = 0.001). Lip licking frequency during the pre- and post-physical examination phases was also significantly lower in the gabapentin treatment group than in the placebo treatment group (P = 0.004). No serious adverse events were reported by the owners following gabapentin treatment. CLINICAL RELEVANCE: Results showed that the 50-mg/kg dose of gabapentin was well tolerated without serious adverse effects in healthy dogs. Further studies are recommended of dogs with documented stress in response to a veterinary visit.

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses.

BACKGROUND: A systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood-derived mesenchymal stromal cells (PB-MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB-MSC administered IV to horses is well-tolerated but therapeutic benefits are unknown. HYPOTHESIS: After IV lipopolysaccharide (LPS) infusion, horses treated with PB-MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo. ANIMALS: Sixteen horses were included in this study. METHODS: A randomized placebo-controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 109 PB-MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24-hour period. RESULTS: A predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB-MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of allogeneic PB-MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB-MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.

Salivary Chromogranin A (CgA) Response to the Noradrenaline Transporter Blocker Atomoxetine in Dogs.

Since salivary chromogranin A (CgA) is one of the known sympathetic adrenomedullar system (SAM) stress markers in humans and pigs, this study aimed to investigate whether salivary CgA in dogs reflects SAM activation. Our hypothesis was that salivary CgA would increase when central noradrenaline was pharmacologically induced. A selective noradrenaline transporter blocker, atomoxetine, was orally administered without causing any aversive responses in nine laboratory dogs to see if it would increase salivary CgA. Three treatment groups (i.e., atomoxetine, placebo, and pre-administration of a selective alpha-2 adrenoreceptor agonist (dexmedetomidine) followed by atomoxetine) were prepared with a randomized crossover design. Saliva sample collection, heart rate measurement and behavior observation were performed at Time 0 (baseline) and at 30, 60, 90 and 150 min after each treatment administration. The results demonstrated that salivary CgA significantly increased at 90 min in the atomoxetine treatment (p < 0.05), whereas it was not observed in the other two treatments. The present study showed that salivary CgA was increased by atomoxetine-induced SAM activation. However, this increase was blocked if dexmedetomidine was pre-administered. Overall, the results indicate that salivary CgA is a potential candidate for SAM-mediated stress markers in dogs. Further study to determine the dynamics of salivary CgA will be helpful in its practical use.

Effects of forages, dust exposure and proresolving lipids on airway inflammation in horses.

OBJECTIVE: To investigate the role of omega-3 polyunsaturated fatty acids (Ω-3)-derived proresolving lipid mediators (PRLM) in the resolution of mild airway inflammation in horses. ANIMALS: 20 horses with mild airway inflammation. PROCEDURES: Horses previously eating hay were fed hay pellets (low Ω-3 content; n = 10) or haylage (high Ω-3 content; 9) for 6 weeks. Dust exposure was measured in the breathing zone with a real-time particulate monitor. Bronchoalveolar lavage (BAL) was performed at baseline, week 3, and week 6. The effect of PRLM on neutrophil apoptosis and efferocytosis was examined in vitro. BAL fluid inflammatory cell proportions, apoptosis of circulating neutrophils, efferocytosis displayed by alveolar macrophages, and plasma lipid concentrations were compared between groups fed low and high amounts of Ω-3 by use of repeated measures of generalized linear models. RESULTS: Dust exposure was significantly higher with hay feeding, compared to haylage and pellets, and equivalent between haylage and pellets. BAL fluid neutrophil proportions decreased significantly in horses fed haylage (baseline, 11.8 ± 2.4%; week 6, 2.5 ± 1.1%) but not pellets (baseline, 12.1 ± 2.3%; week 6, 8.5% ± 1.7%). At week 6, horses eating haylage had significantly lower BAL neutrophil proportions than those eating pellets, and a significantly lower concentration of stearic acid than at baseline. PRLM treatments did not affect neutrophil apoptosis or efferocytosis. CLINICAL RELEVANCE: Despite similar reduction in dust exposure, horses fed haylage displayed greater resolution of airway inflammation than those fed pellets. This improvement was not associated with increased plasma Ω-3 concentrations. Feeding haylage improves airway inflammation beyond that due to reduced dust exposure, though the mechanism remains unclear.

Delivery of drugs and macromolecules to mitochondria.

Mitochondria is where the bulk of the cell's ATP is produced. Mutations occur to genes coding for members of the complexes involved in energy production. Some are a result of damages to nuclear coded genes and others to mitochondrial coded genes. This review describes approaches to bring small molecules, proteins and RNA/DNA into mitochondria. The purpose is to repair damaged genes as well as to interrupt mitochondrial function including energy production, oxygen radical formation and the apoptotic pathway.

Binding of mitochondrial leader sequences to Tom20 assessed using a bacterial two-hybrid system shows that hydrophobic interactions are essential and that some mutated leaders that do not bind Tom20 can still be imported.

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two-hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C-terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1beta-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well ( approximately 50%) while the mutant from F1beta-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.

Factors that might affect the allotopic replacement of a damaged mitochondrial DNA-encoded protein.

The human mitochondrion contains a small circular genome that codes for 13 proteins, 22 tRNAs, and 2 rRNAs. The proteins are all inner membrane bound components of complexes involved in the electron transport system and ATP formation. Mutations to any of the 13 proteins affect cellular behavior because energy production could be decreased. Investigators have attempted to find methods to correct these mutated proteins. One way is to express the mitochondrial gene in the nucleus (called allotopic expression). The newly synthesized protein would have to be imported into mitochondria and assembled into complexes. This paper reviews some of the successful attempts to achieve allotopic expression and discusses some issues that might affect the ability to have the proteins properly inserted into the inner membrane.

A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

Timing and structural consideration for the processing of mitochondrial matrix space proteins by the mitochondrial processing peptidase (MPP).

Most mitochondrial matrix space proteins are synthesized as a precursor protein, and the N-terminal extension of amino acids that served as the leader sequence is removed after import by the action of a metalloprotease called mitochondrial processing peptidase (MPP). The crystal structure of MPP has been solved very recently, and it has been shown that synthetic leader peptides bind with MPP in an extended conformation. However, it is not known how MPP recognizes hundreds of leader peptides with different primary and secondary structures or when during import the leader is removed. Here we took advantage of the fact that the structure of the leader from rat liver aldehyde dehydrogenase has been determined by 2D-NMR to possess two helical portions separated by a three amino acid (RGP) linker. When the linker was deleted, the leader formed one long continuous helix that can target a protein to the matrix space but is not removed by the action of MPP. Repeats of two and three leaders were fused to the precursor protein to determine the stage of import at which processing occurs, if MPP could function as an endo peptidase, and if it would process if the cleavage site was part of a helix. Native or linker deleted constructs were used. Import into isolated yeast mitochondria or processing with recombinantly expressed MPP was performed. It was concluded that processing did not occur as the precursor was just entering the matrix space, but most likely coincided with the folding of the protein. Further, finding that hydrolysis could not take place if the processing site was part of a stable helix is consistent with the crystal structure of MPP. Lastly, it was found that MPP could function at sites as far as 108 residues from the N terminus of the precursor protein, but its ability to process decreases exponentially as the distance increases.

In vitro evidence of inhibition of mitochondrial protease processing by HIV-1 protease inhibitors in yeast: a possible contribution to lipodystrophy syndrome.

Highly active antiretroviral therapy has been associated with the emergence of lipodystrophy syndromes that have clinical features commonly seen in patients with mitochondrial dysfunction. The effect of therapeutic protease inhibitors (PIs) on mitochondrial function is unknown. Mitochondrial matrix space proteins possess an amino-terminal leader peptide that is removed by the mitochondrial processing protease (MPP). Lack of cleavage could result in non- or dysfunctional mitochondrial proteins. The effects of different PIs on protease processing using pure MPP or yeast mitochondria, recognized models for mammalian counterparts, were examined in vitro. Multiple PIs were found to inhibit MPP, evidenced by accumulation of immature pALDH and decreased levels of processed ALDH. Both indinavir and amprenavir at 5.0 mg/ml resulted in significant inhibition of MPP. Although inhibition of MPP was also observed with ritonavir and saquinavir, the inhibition was difficult to quantify due to background inhibition of MPP by DMSO that was required to solubilize the drugs for the in vitro studies. Indinavir was also shown to inhibit MPP within yeast mitochondria. Lack of processing may impair mitochondrial function and contribute to the observed mitochondrial dysfunctions in patients receiving HAART and implicated in antiretroviral-associated lipodystrophy.

A point mutation produced a class 3 aldehyde dehydrogenase with increased protective ability against the killing effect of cyclophosphamide.

Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.

Precursor protein is readily degraded in mitochondrial matrix space if the leader is not processed by mitochondrial processing peptidase.

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins.

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.

Location of the actual signal in the negatively charged leader sequence involved in the import into the mitochondrial matrix space.

Proteins destined for the mitochondrial matrix space have leader sequences that are typically present at the most N-terminal end of the nuclear-encoded precursor protein. The leaders are rich in positive charges and usually deficient of negative charges. This observation led to the acid-chain hypothesis to explain how the leader sequences interact with negatively charged receptor proteins. Here we show using both chimeric leaders and one from isopropyl malate synthase that possesses a negative charge that the leader need not be at the very N terminus of the precursor. Experiments were performed with modified non-functioning leader sequences fused to either the native or a non-functioning leader of aldehyde dehydrogenase so that an internal leader sequence could exist. The internal leader is sufficient for the import of the modified precursor protein. It appears that this leader still needs to form an amphipathic helix just like the normal N-terminal leaders do. This internal leader could function even if the most N-terminal portion contained negative charges in the first 7-11 residues. If the first 11 residues were deleted from isopropyl malate synthase, the resulting protein was imported more successfully than the native protein. It appears that precursors that carry negatively charged leaders use an internal signal sequence to compensate for the non-functional segment at the most N-terminal portion of the protein.